ITS2 secondary structure for species circumscription: case study in southern African Strychnos L. (Loganiaceae).

Recently developed computational tools in ITS2 sequence-structure phylogenetics are improving tree robustness by exploitation of the added information content of the secondary structure. Despite this strength, however, their adoption for species-level clarifications in angiosperms has been slow. We investigate the utility of combining ITS2 sequence and secondary structure to separate species of southern African Strychnos, and assess correlation between compensatory base changes (CBCs) and currently recognised species boundaries. Combined phylogenetic analysis of sequence and secondary structure datasets performed better, in terms of robustness and species resolution, than analysis involving primary sequences only, achieving 100 and 88.2 % taxa discriminations respectively. Further, the Strychnos madagascariensis complex is well-resolved by sequence-structure phylogenetic analysis. The 17 Strychnos species corresponded to 14 ITS2 CBC clades. Four of the five taxa in section Densiflorae belong to a single CBC clade, whose members tend to form natural hybrids. Our finding supports the application of ITS2 as a complementary barcoding marker for species identification. It also highlights the potential of comparative studies of ITS2 CBC features among prospective parental pairs in breeding experiments as a rapid proxy for cross compatibility assessment. This could save valuable time in crop improvement. Patterns of CBC evolution and species boundaries in Strychnos suggests a positive correlation. We conclude that the CBC pattern coupled with observed ITS2 sequence paraphyly in section Densiflorae points to a speciation work-in-progress.

A simple method to identify the unprocessed Strychnos seeds used in herbal medicinal products.

The seeds of Strychnos nux-vomica are popularly used in the treatment of arthritis. Being extremely toxic, the raw seeds are forbidden and must be processed before clinical use. The quality of crude and processed Strychnos seeds can be controlled by examining the toxic alkaloids using established HPLC methods. But this procedure does not work in the case where the seeds are powdered and mixed with other medicinal materials in proprietary production. In this quality control study on Strychnos seeds, the contents of two major toxic alkaloids (strychnine and brucine) and a major non-alkaloid constituent (loganic acid) in twenty-four samples of Strychnos seeds (nine processed and fifteen unprocessed) were compared using published HPLC-UV methods. The results showed that the better the seeds were processed, the less loganic acid was found. The alkaloids and non-alkaloid components simultaneously decreased in processed seeds. The content ratio between alkaloids and loganic acid was clearly different in well-processed and crude Strychnos seeds. Based on this interesting discovery, a simple chromatographic method was established which allows a simultaneous determination of loganic acid and the alkaloids strychnine and brucine. The relative peak area (RPA) of strychnine-to-loganic acid was revealed to be a reliable key quality control parameter in order to effectively identify the processed seeds. This new method has been successfully applied to detect the insufficiently processed Strychnos material in marketed herbal medicinal products.

Metabolomic analysis of Strychnos nux-vomica, Strychnos icaja and Strychnos ignatii extracts by 1H nuclear magnetic resonance spectrometry and multivariate analysis techniques.

1H Nuclear magnetic resonance spectrometry and multivariate analysis techniques were applied for the metabolic profiling of three Strychnos species: Strychnos nux-vomica (seeds, stem bark, root bark), Strychnos ignatii (seeds), and Strychnos icaja (leaves, stem bark, root bark, collar bark). The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between all samples, using the three first components. The key compounds responsible for the discrimination were brucine, loganin, fatty acids, and Strychnos icaja alkaloids such as icajine and sungucine. The method was then applied to the classification of several "false angostura" samples. These samples were, as expected, identified as S. nux-vomica by PCA, but could not be clearly discriminated as root bark or stem bark samples after further statistical analysis.